The present study was conducted to develop an effective system for in vitro fertilization of pig oocytes. A series of experiments had been done to examine the effects of various factors during culture of oocytes on normal cytoplasmic maturation that leads normal male pronuclear formation or monospermy after fertilization in vitro. It was found that epidermal growth factor and/or gonadotropins (eCG+hCG) can stimulate oocyte maturation in serum-free medium and this stimulative effect is mediated by the presence of cumulus cells. When cumulus-enclosed oocytes were cultured in BSA-free Whitten's medium (mWM) and modified TCM-199 (TCM-199B), cytoplasmic maturation of oocytes was affected by gonadotropins and/or fetal calf serum and/or pig follicular fluid (PFF) in different ways in different media. Further experiments were conducted to examine the ability of oocytes to achieve male pronuclear formation when they are matured and penetrated in vitro under various culture conditions. The resul
… Morets indicated that male pronuclear formation in oocytes is promoted by the addition of 20 amino acids and/or cysteine in simple maturation medium (mWM supplemented with gonadotropins and PFF) and by the presence of cumulus cells at fertilization in vitro. It was also clarified that the presence of cysteine in serum-free maturation medium is required only between 42 and 48 h after initiation of maturation for promotion of male pronuclear formation after sperm penetration in vitro. Male pronuclei can be formed in cumulus-free oocytes matured in the presence of cysteine, but the presence of cumulus cells is essential for maintainingmale pronuclear formation in oocytes matured in the presence of cystine. In the study examined penetration of immature oocytes, it was demonstrated that the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes. Although further improvements in the conditions used for in vitro maturation and fertilization are needed, the results obtained in the present study would contribute to ensure the production of larger numbers of normal pig embryos.1.未熟卵子の細胞質成熟におよぼす上皮成長因子(EGF)と性腺刺激ホルモン(GTH;10IU eCG/ml+10IU bCG/ml)の相乗効果について調べ、EGFとGTHはいずれも細胞質成熟を促進したが、この効果は卵丘細胞を介して発現されることを明らかにした。2.さらに培養条件を検討するために、異なる培地で培養した未熟卵子の成熟におよぼす牛胎児血清(FCS)、豚卵胞液(PFF)およびGTHの影響について調べた。成熟培地としてTCM-199BおよびWhittenの修正培地(mWM)を用いて比較した結果、異なる培地においてGT、FCSおよびPFFは卵子の成熟それぞれ異なる影響をおよぼすことが示唆された。また、PFFおよびGTを添加したmWM(mWM-FG)にさらに20種類のアミノ酸を添加した結果、細胞質成熟はより促進されるとともに、受精時に卵丘細胞が存在することによって雄性前核形成の促進されることを明らかにした。3.システイン(CT)の細胞質成熟促進効果についてはすでに知られているが、その効果の発現が卵子の成熟段階に依存していることを明らかにした。また、CTとシスチンの卵子による利用性が卵丘細胞の有無により異なることを示唆する結果を得た。4.第一成熟分裂途上の未熟卵子への精子侵入能の変化と細胞質内に侵入した精子核の変化について調べ、第一成熟分裂途上の未熟卵子の細胞質が精子の中期像染色体を形成する強い活性を有することを見いだした。 Less